The transgenic mouse expresses a mutant ezrin gene in which the C-terminal phosphorylation site is mutated to an acidic residue to mimic constitutive phosphorylation (T567E) and tagged with a short peptide HA tag at the extreme C-terminus. The gene is selectively expressed in T-cells (by use of a CD2 cassette) and protein expression is at most half of the endogenous ezrin levels. Studies of this mouse are ongoing and include comparisons with a transgenic mouse strain expressing a comparable construct without the mutation. First, we needed to assess overall immune system development. Our studies indicate that it is close to normal in heterozygous transgenic mice. For example, there are small changes in parameters describing T-cell populations, but all the changes are so limited that they become statistically significant only with large sample number: e.g. decreases of about 10% in T-cell number in lymph nodes, in peripheral CD4/CD8 ratio and in % of nave cells. Similarly, these mice mount normal IgG1 and IgG2a antibody responses to TNP-KLH with alum and there is a statistically insignificant decrease in their response in the absence of adjuvant. Second, we are investigating our central hypothesis that presence of constitutively active ERM impairs shape change and migration. We have initiated collaborative studies of migration in lymph node using two-photon microscopy and analysis with Imaris.. Results of pilot studies show a profound change in migration. The mean migration speed is reduced 20-50 percent in the T567E transgenic cells (Fig 2). The histogram of individual cell migration speed is broad for lymphocytes from each strain and generally shifted towards slower migration speed in the transgenic. In addition, directional persistence appears to be impaired since straightness of migration is also significantly reduced in the transgenic lymphocytes. Thus, these studies are confirming that the dominant active T567E ezrin protein profoundly alters in vivo migration. Membrane tension of the plasma membrane is biophysical process that is viewed as a master regulator of many cellular processes, but has not been studied in lymphocytes. We hypothesized that phospho-ERM would increase membrane tension, because of its expected increase in linkage between plasma membrane and cortical actin. Moreover, such an increase in membrane tension could be an important contributor to the impaired migration. Analysis of pulled membrane tethers reveals about a 50% increase in membrane tension in T567E transgenic T-cells.